Erythropoietin signaling mediated by the cytokine binding to its receptor is required for erythroid progenitor cells survival, proliferation and differentiation. However, erythropoietin signaling is not restricted to the erythroid lineage. Importantly, some metabolic effects of erythropoietin including protection against diet induced obesity and improvement of insulin resistance and glucose tolerance were recently reported. The skeletal muscle rich in slow twitch fiber expresses more genes related to oxidative function and mitochondrial activity compared to fast twitch fiber rich muscle. We examined the impact of erythropoietin signaling on the muscle fiber development, mediated in part via expression of the erythropoietin receptor in myoblasts. Our data showed that there are more slow twitch myofibers phenotype and mitochondrial activity in mice skeletal muscle with transgenic overexpression of erythropoietin compared to wide type mice but less slow twitch myofibers and mitochondrial activity in non-hematopoietic specific knock out of erythropoietin receptor. Importantly, PGC-1a, which activates mitochondrial biogenesis and oxidative metabolism and converts the fast myofibers to slow myofibers when overexpressed in skeletal muscle, was found elevated in erythropoietin overexpressed mice. Our in vitro experiments demonstrated that erythropoietin can increase mitochondrial biogenesis genes expression, oxidative activity and PGC-1a gene expression in skeletal muscle satellite cells and myoblasts. Our data also confirmed that erythropoietin can activate AMPK activity and under hypoxia condition, AMPK activity can be stimulated longer by erythropoietin. Taken together, these data suggest that erythropoietin can be involved the skeletal muscle fiber programming and muscle metabolism possibly via increased PGC-1a and AMPK activity during muscle development.